Antimicrobial Susceptibility and Molecular Characterization of Carbapenemase-Producing Enterobacter spp. Community Isolates in Belgrade,Serbia

Introduction

The prevalence of carbapenem-resistant Enterobacterales has increased over the last decade and it represents one of the major problems of modern medicine. ¹ Resistance to carbapenems is mediated by β-lactamase activity, in combination with structural mutations that lead to membrane impermeability, or by production of carbapenemases.^2,3 Carbapenemase genes are located mostly on plasmids that often harbor genes resistant to other antimicrobial agents, and these multidrug-resistant clones spread rapidly worldwide. ³

After the first report of carbapenemase-producing (CP) Enterobacterales in Japan more than two decades ago, ⁴ they have been detected in all inhabited continents, with defined endemic areas. ⁵ Several studies have shown that Balkan countries are reservoirs of carbapenemases, especially bla_NDM-1.^6–9 A recent European Survey of Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) categorized Serbia as one of the countries with the highest prevalence rates of CP isolates of Klebsiella pneumoniae and Escherichia coli. ¹⁰ Despite the fact that Enterobacter spp. is one of the three most common CP Enterobacterales, molecular epidemiology data for this genus are limited. ⁹ Furthermore, the majority of reports refer to nosocomially acquired carbapenemases, but data from the community are unknown or underrepresented.^11–13

Therefore, the aims of the present study were to estimate the prevalence of carbapenemase-producing (CP) Enterobacter spp. among community isolates in Belgrade, to identify the types of carbapenemases, and to determine genetic relatedness of these isolates.

Materials and Methods

Results

During the study period, a total of 1,040 Enterobacter spp. isolates from different clinical samples were screened for carbapenemase production: 795 (76.4%) were obtained from CIPH and 245 (23.6%) from ILDK. Most of the isolates originated from superficial wound swabs (36.6%, 380/1,040) and urine samples (34.2%, 356/1,040). Other specimens (29.2%, 304/1,040) included sputum, ear swabs, and eye swabs.

Screening for carbapenemase production was positive for 17 (1.6%) strains, all isolated from urine samples and identified by MALDI-TOF MS as Enterobacter cloacae/asburiae. Ten (58.8%) isolates were obtained from ILDK and 7 (41.2%) from CIPH.

All isolates were multidrug resistant with acquired resistance to piperacillin–tazobactam, ceftriaxone, ceftazidime, cefepime, ertapenem, ciprofloxacin, levofloxacin, and gentamicin. High rates of nonsusceptibility (including both intermediate resistant and resistant isolates) to trimethoprim–sulfamethoxazole (94.1%), chloramphenicol (82.3%), amikacin (70.6%), and tigecycline (70.6%) were detected. All tested isolates were susceptible to colistin (MICs: ≤0.25–0.5 μg/mL, Table 1).

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Table 1.

Minimum Inhibitory Concentrations, Distribution of Carbapenemase Genes, and Enterobacterial Repetitive Intergenic Consensus–Polymerase Chain Reaction Clusters of Carbapenemase-Producing Enterobacter cloacae/asburiae Community Isolates

Carbapenemase-encoding genes were detected in all carbapenemase screening-positive E. cloacae/asburiae isolates, with 70.6% (12/17) of them harboring bla_NDM, 23.5% (4/17) carrying both bla_NDM and bla_OXA-48-like, and one isolate (5.9%) harboring the bla_OXA-48-like gene. A higher incidence of bla_NDM-bla_OXA48-like was detected in isolates obtained from ILDK (p < 0.05). Antimicrobial susceptibility and carbapenemase gene distribution are summarized in Table 1.

Molecular typing of the 17 CP E. cloacae/asburiae community isolates showed distribution in six clusters (A–F), three with a single isolate and three clusters with more than one isolate (Table 1, Fig. 1). A significant difference in frequency of clusters was noticed between isolates obtained from the different institutes: a higher frequency of cluster F was found in isolates from ILDK (p < 0.05) and a higher frequency of cluster E in isolates from CIPH (p < 0.05). Higher frequency of carbapenemase-encoding genes bla_NDM-bla_OXA48-like was associated with cluster F (p < 0.05) and higher frequency of bla_NDM in E and F clusters, compared with all other clusters (p < 0.05).

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FIG. 1.

Dendrogram and gel image obtained by enterobacterial repetitive intergenic consensus–polymerase chain reaction analysis of carbapenemase-producing Enterobacter cloacae/asburiae community isolates (n = 17). Six different clusters (A–F) were generated at the similarity level of 90% among isolates from CIPH and ILDK. CIPH, City Institute of Public Health of Belgrade; ILDK, Institute for Laboratory Diagnostics “Konzilijum” Belgrade.

Discussion

Several international studies and surveillance programs have observed a high rate of antimicrobial resistance (AMR) in invasive isolates of K. pneumoniae and Escherichia coli in Serbia,^10,13 as well as one national investigation of hospital isolates of these bacteria. ²⁵ To the best of our knowledge, there have been no AMR data on Enterobacter spp. in Serbia. In the present study, we determined the prevalence (1.6%) of CP E. cloacae/asburiae among community isolates in the Belgrade region. The overall number of studies based in community settings is limited. In a Belgian multicentric study, no CP Enterobacter spp. were found among isolates from community outpatients. ²⁶ A similar Australian community-based survey reported only one carbapenemase-positive isolate of 239 Enterobacter spp. isolates. ²⁷

A relatively high level of CP E. cloacae/asburiae community isolates in Serbia, all originating from urine samples, could be a consequence of overuse or misuse of antibiotics in treatment of urinary tract infections since these infections represent the main cause of antibiotic prescription and consumption in primary health care settings.^28,29 However, results of our study could be in disagreement with the previous reports due to differences in study population, antimicrobial susceptibility testing methods, and carbapenem breakpoints.

The multiresistant phenotype is almost systematically associated with production of bla_NDM carbapenemases,^30,31 the most common carbapenemase found in Serbian E. cloacae/asburiae isolates. Our result is in accordance with findings of several case reports and studies from Serbia.^32–35 Additionally, a single isolate harboring the bla_OXA-48-like gene and four isolates carrying both bla_NDM and bla_OXA-48-like carbapenemase genes were detected. These findings represent the first report of the bla_OXA-48-like-positive isolate of Enterobacter spp. in Serbia and of coexistence of the bla_NDM and bla_OXA-48-like genes.

It is believed that bla_OXA-48-like carbapenemases originated in Turkey and have spread dominantly in Mediterranean countries. ³¹ Taking into account that Balkan countries lie on the major migration route from the Middle East and North Africa to Europe, dissemination of bla_OXA-48-like genes and their occurrence in Serbia are expected. This is supported by results of the molecular epidemiology study on global collection of Enterobacter spp., which showed that bla_NDM-positive isolates from Serbia and bla_OXA-48-positive isolates from Middle East (Tunisia, Morocco, and Kuwait) belong to the same ST114A clade. ⁹

Dissemination of carbapenem-resistant E. cloacae/asburiae is associated with high clonal diversity accompanied by a variety of carbapenemase genes, e.g., there is no specific clone determined for bla_NDM-harboring isolates on a global level.^9,30,31 Annavajhala et al. emphasized underreporting of carbapenem-resistant E. cloacae/asburiae as one of the reasons for gaps in understanding its diversity. ³¹ Contrary to these results, Peirano et al. have shown local spreading of certain clones and clades, with specific genetic elements and carbapenemase genes. ⁹ In accordance with the last, a phylogenetic analysis of Serbian community E. cloacae/asburiae isolates showed clonal relatedness of the isolates from the same institution. These locally circulating clones were associated with the specific carbapenemase gene, bla_NDM. Furthermore, cluster F has shown a higher frequency of isolates harboring both bla_NDM and bla_OXA48-like genes.

However, the major limitation of our study was the lack of clinical epidemiological data that would be of great value for analyzing the route of carbapenem-resistant E. cloacae/asburiae dissemination in community settings and from other countries. Since organization of the epidemiological survey was not feasible for a large number of patients, our study was designed as a cross-sectional study for preliminary screening of the epidemiological situation. Our results need further detailed population-level analysis with more specific typing methods.

In summary, our study represents the first analysis of Enterobacter spp. isolates in Serbia. Multidrug resistance and carbapenemase production with clonal dissemination observed among community isolates in Belgrade, Serbia, indicate a clear future need for carbapenemase surveillance of Enterobacter spp., both in community and hospital settings.